The berkeleyamides, amides from the acid lake fungus Penicillum rubrum

Article abstract of DOI:10.1021/np0705054

We previously reported several novel bioactive hybrid polyketide-terpenoid metabolites from a deep water Penicillium rubrum isolated from Berkeley Pit Lake, Butte, Montana. In this paper we report the structures of four new amides, berkeleyamides A - D (1, 4, 5, 7), isolated from extracts of this fungus. The structures of these compounds were deduced by analysis of NMR data, chemical derivatization, and comparison of their spectroscopic data to those of known compounds.

Full text of DOI:10.1021/np0705054

856
J. Nat. Prod. 2008, 71, 856–860
The Berkeleyamides, Amides from the Acid Lake Fungus Penicillum rubrum
Andrea A. Stierle,* Donald B. Stierle, and Brianna Patacini
Department of Chemistry and Geochemistry, Montana Tech of the UniVersity of Montana, Butte, Montana 59701
ReceiVed September 20, 2007
We previously reported several novel bioactive hybrid polyketide-terpenoid metabolites from a deep water Penicillium
rubrum isolated from Berkeley Pit Lake, Butte, Montana. In this paper we report the structures of four new amides,
berkeleyamides A-D (1, 4, 5, 7), isolated from extracts of this fungus. The structures of these compounds were deduced
by analysis of NMR data, chemical derivatization, and comparison of their spectroscopic data to those of known
compounds.
The Berkeley Pit Lake System is part of the largest EPA
Superfund site in North America.1 We have isolated over 70 unique
microbes from this evolving extreme ecosystem and are in the
process of studying the bioactive secondary metabolites as possible
anticancer or antimicrobial agents. Several interesting compounds
have been isolated from these microbes on the basis of their
activities in different bioassay-guided fractionation schemes.2–6
One of the first microbes to be studied from this environment
was isolated from a water sample taken from a depth of 885 feet
and identified as Penicillium rubrum Stoll.3,7 The organic extracts
of this fungus inhibited the signal transducing enzymes matrix
metalloproteinase-3 and caspase-1. We have previously reported
several novel bioactive compounds isolated from the extracts of
this fungus: the berkeleyacetals,7 berkeleydione, and berkeleytrione,
all of which inhibited MMP-3 and caspase-1.3
The berkeleyamides were also isolated on the basis of inhibition
of MMP-3 and caspase-1. MMP-3 is up-regulated in many
tumors.8,9 Specific MMP inhibitors block the activity of proteolytic
CH) and 127.2(CH)), four methylene carbons (δ 50.6, 46.2, 46.0,
enzymes (MMPs) used by tumor cells to promote metastatic
spread.10,11 Caspase-1 inhibitors have shown promise in halting
tumor progression, delaying the onset of Huntington’s disease12
and amyotropic lateral sclerosis,13 mitigating the effects of stroke14
and multiple sclerosis,15,16 and inhibiting the proliferation of acute
myelogenous leukemia (AML) progenitor cells.17 Specific caspase-1
inhibitors might provide a new class of antileukemia or anti-
inflammatory drugs with multipotent action.18 Inhibition of these
two enzymes guided isolation of the berkeleyamides.
The fungus was grown in liquid cultures using acidified potato
dextrose broth (pH 2.7) for 21 days. At harvest time the fungus
was killed with the addition of MeOH. The culture was filtered
through cheesecloth to remove the mycelial mat. The filtrate was
extracted with CHCl3, and the extract was reduced in Vacuo to an
oil. The CHCl3 extract inhibited both MMP-3 and caspase-1 in the
assay systems in the micromolar range. It was fractionated by flash
silica gel column chromatography followed by HPLC to yield
berkeleyamides 1, 4, 5, and 7.
and 28.7), four methines (δ 66.8, 50.5, 45.3, and 25.2), and two
methyl carbons (δ 22.9 and 22.2). The benzene ring and two
carbonyl functionalities accommodated six sites of unsaturation and
indicated the presence of an additional ring. The 1H NMR spectrum
clearly showed five aromatic protons (δ 7.28–7.16m), an isolated
methylene group (δ 3.72), and two overlapping methyl doublets.
Analysis of the COSY spectrum indicated the presence of three
discrete spin systems: a monosubstituted benzene ring, an isolated
methylene (H2-7, δ 3.72), and an extended system of protons
attached to sp3-hybridized carbons: [-CH2-CH-CH-CH2-CH
-CH2-CH(CH3)2]. Careful analysis of the data provided a clear
stepwise path along the backbone connecting methylene H2-9 (δ
2.83, 2.68) to H-10 (δ 4.36), which was spin-coupled to methine
H-11 (δ 2.44). H-11 was coupled to methylene H2-19 (δ 2.29, 1.69),
which coupled to methine H-14 (δ 3.62), which coupled to
methylene H2-15 (δ 1.28). H2-15 was finally coupled to a terminal
isopropyl moiety, H-16–H-18.
Examination of mass spectra, IR, 1H NMR, 1H-1H COSY,
HSQC, and HMBC spectra provided the necessary information to
determine the structures and the relative configurations of the
berkeleyamides. HRESIMS established a molecular formula of
C18H25NO3 for berkeleyamide A (1), with seven double-bond
equivalents (DBE). The IR spectrum indicated either an OH or NH
stretch and carbonyl stretching frequencies indicative of both a
ketone (1715 cm-1) and an amide (1685 cm-1). The 13C NMR and
DEPT spectra (see Table 1) also provided support for the presence
of the ketone (δ 208.8) and amide (δ 177.2), as well as a
monosubstituted aromatic ring (δ 133.5, 129.5 (2C, CH), 128.8 (2C,
The HMBC spectrum provided several key correlations that
supported this partial structure and generated the complete carbon
backbone of berkeleyamide A. Long-range correlations from
isolated methylene protons H2-7 to aromatic carbons C-1, C-5 (δ
129.5, 2C), and C-6 (δ 133.5) and to ketone carbon C-8 (δ 208.8)
indicated its benzylic position. The terminal methylene of the
extended spin system H2-9 also showed long-range correlations to
the ketone carbon, which bridged these two spin systems. Both
carbon and proton chemical shifts indicated that C-10 was oxygen
bearing (δC 66.8, δH 4.36) and that C-14 was nitrogen bearing (δC
50.5, δH 3.62). Acetylation of 1 gave a monoacetate (2), which
showed the expected downfield shift of H-10 from δ 4.36 to δ 5.48.
In addition, methine H-11 showed HMBC correlations to both
amide C-12 and oxygen-bearing methine C-10, and the NH proton
* To whom correspondence should be addressed. Tel: (406) 496-4717.
Fax: (406) 496-4135. E-mail: astierle@mtech.edu.
10.1021/np0705054 CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 03/11/2008

Notes
Journal of Natural Products, 2008, Vol. 71, No. 5 857
a
1
Table 1. 13C and H NMR Data for Berkeleyamides A (1), B (4), C (5), and D (7) in CDCl3
1
4
5
7
no.
1, 5
2, 4
3
6
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
129.5 7.28 m
128.8 7.16 m
127.2 7.16 m
133.5
129.2 7.38 m
127.4 7.38 m
129.8 7.38 m
134.2
129.4 7.38 m
127.9 7.28 m
129.9 7.28 m
134.5
129.2 7.33 m
129.0 7.33 m
127.0 7.33 m
133.2
7
8
50.6 3.72 s, 2H
208.8
72.4 6.54 s
166.4
71.4 6.83 s
149.8
37.4 3.98 d (17.4), 3.96 d (17.4)
197.8
9
10
11
12
46.2 2.83 dd (17.4, 3.1) 2.68 dd (17.4, 9.0)
66.8 4.36 ddd (9.0, 5.3, 3.1)
45.3 2.44 ddd (9.3, 7.2, 5.3)
177.2
114.8 6.59 s
177.6
119.3
120.6 6.85 s
177.4
117.1
104.4 5.35 bs
199.4
95.3
160.4
163.2
164.1
13
6.02 bs
11.53 bs
13.22 bs
6.78 bs
14
15
16
17
18
19
Ac
50.5 3.62 qd (7.4, 4.1)
46.0 1.37 m, 2H
25.2 1.57 m
22.9 0.88 d, 6H (6.6)
22.2 0.88 d, 6H (6.6)
28.7 ꢀ2.29 dt (12.8, 7.6) R1.69 ddd (12.9, 9.1, 4.1) 163.0 8.69 bs
173.3
173.2
84.9
47.3 2.58 d, 2H (6.9)
24.9 2.17 m
22.4 0.96 d, 6H (6.7)
22.4 0.96 d, 6H (6.7)
47.8 2.32 d, 2H (7.0)
25.6 2.17 m
22.4 0.97 d, 6H (6.6)
22.4 0.97 d, 6H (6.6)
150.4 8.97 bs
169.4
45.5 1.88 m, 2H
24.0 1.92 m
23.9 1.00 d, 3H (5.2)
23.8 0.98 d, 3H (5.2)
75.1 4.41 d (10.0)
169.0
Ac-Me
20
21
20.7 2.18 s, 3H
20.9 2.17 s, 3H
56.1 4.11 m, 2H
61.1 3.85 m, 2H
OH,C-19
OH,C-14
3.04 d (10.0)
5.48 bs
a All assignments are based on COSY, NOE, HSQC, and HMBC experiments.
Figure 2. Important HMBC correlations in 4.
an absorption at 1697 cm-1, and a series of absorption frequencies
(1654, 1616, and 1574 cm-1) that suggested the presence of a
4-pyrone.20
Figure 1. Selected ∆δ values around C-10 of the (R) and (S)-MTPA
esters of compound 3 [∆δ ) chemical shift of (R)-MTPA ester
minus chemical shift of (S)-MTPA ester in ppm].
The structure of 4 was established by consideration of these data
and extensive analysis of the HMBC spectrum. The most important
long-range correlations are shown in Figure 2. The unusually
deshielded methine proton H-7 (δH 6.54) attached to oxygen-bearing
C-7 (δ 72.4) showed HMBC correlations to the acetate carbonyl,
to aromatic carbons C-6, C-1/C-5, and C-2/C-4, and to olefinic
carbons C-8 (δ 166.4) and C-9 (δ 114.8). These connections helped
explain the downfield shift of H-7. Olefinic proton H-9 (δ 6.59)
also showed correlations to methine C-7, to carbonyl C-10 (δ
177.6), and to olefinic carbons C-11 and C-19 (δ 119.3, 163.0,
respectively). These last two carbons showed HMBC correlations
to olefinic proton H-19 (δ 8.69). These HMBC correlations were
useful in generating a disubstituted 4-pyrone ring. The NMR
chemical shifts of the disubstituted pyrone ring compared favorably
with those of the 2,5-disubstituted-4-pyrone, microsphaerone A
(S10, Supporting Information), isolated from the fungus Mi-
crosphaeropsis sp.21
showed correlations to amide C-12 and to nitrogen-bearing C-14.
It remained to connect N-13 to C-12 to generate a γ-lactam and
the proposed structure for 1.
Establishing the overall configuration of this compound proved
more challenging than was anticipated. The relative configuration
of the amide ring was established by 1D NOE difference spectros-
copy. Irradiation of H-14 enhanced both Hꢀ-19 (δ 2.29) and H-13.
Irradiation of H-11 enhanced HR-19 (δ 1.69) and H-10 (4.6%),
which clearly established the trans relationship of H-11 and H-14.
We attempted to determine the absolute configuration of berke-
leyamide A (1) using a modified Mosher method.19 Treatment of
1 with (R)- and (S)-methoxy(trifluoromethyl)phenylacetyl (MTPA)
chloride in pyridine gave the corresponding R- or S-esters respec-
tively (3a, 3b). Molecular modeling of the esters and consideration
of the δ∆ values (see Figure 1) indicated that the absolute
configuration at C-10 was S. However, molecular modeling studies
of the two possible diastereomers did not provide sufficient evidence
to determine unambiguously if the overall structure was (10S),
(11R),(14S) or (10S),(11S),(14R).
Olefinic proton H-19 also showed HMBC correlation to amide
carbonyl C-12 (δ 160.4), whose chemical shift was indicative of
R,ꢀ-unsaturation. The amide proton (δ 11.53) showed long-range
correlations to both amide carbons C-12 and C-14 (δ 160.4, 173.3),
which generated an imide functionality. The NMR absorbances of
the 4-pyrone-imide moiety also compared favorably to those
reported for himeic acid A, a compound isolated from a marine-
derived Aspergillus sp.22 The C-14 resonance showed correlations
to methylene H2-15 (δ 2.58) and methine H-16 (δ 2.17), which
coupled to both methyl H-17 and H-18, generating the isobutyl
end group typical of these compounds. These data generated the
structure proposed for berkeleyamide B.
HRESIMS gave a molecular formula of C20H21NO6 for berke-
leyamide B (4), with 11 sites of unsaturation and two more carbons
than 1. The proton NMR spectrum of berkeleyamide B exhibited
resonances for both the monosubstituted aromatic ring and the
terminal isobutyl moiety found in berkeleyamide A (1). A prelimi-
nary look at the data showed that 14 of the 20 carbons were sp2
hybridized and that the two additional carbons in 4 could be
assigned to an acetate moiety (δC 169.0, 20.7 and δH 2.18 s, 3H).
The IR spectrum supported the presence of an acetate moiety with
a carbonyl absorption at 1754 cm-1, an amide functionality with
HRESIMS gave a molecular formula of C22H26N2O6 for berke-
leyamide C (5) with 11 sites of unsaturation. This formula contained

858 Journal of Natural Products, 2008, Vol. 71, No. 5
Notes
two more carbons, one more nitrogen, and five more hydrogens
1
than 4. The H and 13C NMR (Table 1) spectra for compounds 4
and 5 were very similar, although the NMR spectra of 5 showed
two additional coupled methylenes (δC 61.1, 56.1 and δH 3.85 and
4.11, respectively). In the 13C NMR spectrum, the olefinic carbons
exhibited less extreme shift variations, suggesting that the ring was
a 4-pyridone rather than a 4-pyrone. These differences from 4 could
be accommodated by an N-ethyl-2-hydroxy 4-pyridone ring in 5.
The IR data provided additional support for this assignment: the
absorbance at 1634 cm-1 is typical of the carbonyl stretch of
4-pyridone rings.23
Figure 3. Important HMBC correlations in 7.
was treated with N-trimethylsilylimidazole (TMSIM), a very active
trimethylsilyl donor, the ESIMS spectrum gave a parent ion for
the addition of three trimethylsilyl (TMS) groups, indicating three
OH/NH groups, or enolizable systems. When 7 was treated with
the less active silylating agent N,O-bis(trimethylsilyl)trifluoroac-
etamide (BSTFA), ESIMS indicated the addition of only two TMS.
This was strong evidence that 7 had three silylizable groups, but
only two were the more easily silylated hydroxy groups.25 Further
evidence for the proposed novel azaspirocyclic ring system in 7
was obtained by comparison of the NMR data to those of other
known fungal metabolites. The pseurotins A-F, isolated from
fungal strains of Pseudeurotium oValis,26 and the more recently
reported azaspirene, isolated from the fungus Neosartorya sp.,27
contain a similar azaspirocyclic system. The carbon and proton
chemical shifts around the lactam ring in these compounds are very
close to those of the lactam ring in 7. However, as the furanone
ring in these latter compounds is alkylated, the chemical shifts of
this ring are a bit different. Therefore, the furanone ring shifts were
compared to the furanone ring system of phelligridin E isolated
from the fungus Phellinus igniarius.28 The NMR data of these two
systems compared nicely (S12, Supporting Information).
The presumed biosynthetic building blocks for the pseurotins
and azaspirene are a polyacetate precursor and phenylalanine.29 If
this biosynthetic scheme is used as a model, berkeleyamide D (7)
could be produced from a phenyl triketide precursor and leucine
as shown in Scheme 1. When this fungus was grown with 15N-
labeled leucine, the ESIMS of 7 showed a 2% incorporation of
15N, as evidenced by the increase of the M + 1 peak of the parent
ion [M + Na]+.
Consideration of the structures of berkeleyamides 1, 4, and 5
indicated that they could come from similar biosynthetic precursors.
Conjugation of the phenyl triketide and leucine and reduction of
the carbonyl at C-10 followed by cyclization could give 1 directly.
The biosynthesis of 4 and 5 from these two precursors is a little
more convoluted. The origin of C-19 in all of the berkeleyamides
is of special interest. It could come from the carbonyl carbon of
the leucine group through a hypothetical cyclic intermediate. C-19
could also come from methylation by S-adenosylmethionine, which
is common in polyketide metabolism and is observed in pseurotin
biosynthesis.29
All of these compounds were isolated on the basis of their activity
in enzyme inhibition assays. All four compounds were active against
both caspase-1 and MMP-3 in the low micromolar range. Berke-
leyamide A (1) and berkeleyamide D (7) exhibited the greatest
potency, with IC50 values of 0.33 and 0.61 µM, respectively. These
compounds were submitted to the NCI/NIH Developmental Thera-
peutics Program for testing against their suite of 60 human cancer
cell lines. Although the compounds were accepted for the single-
dose assay, they did not meet the criterion for inclusion in the five-
dose screen.
The long-range correlations in the HMBC of compound 5 were
in full agreement with the proposed structure. In addition to all of
the correlations seen in 4, HMBC correlations from H2-20 (δ 4.11)
to the ꢀ-carbons of the pyridone ring were observed (δ 149.8,
150.4). Acetylation of 5 gave the monoacetate 6 with the expected
downfield shift of the methylene protons H2-21 (δ 3.85 to 4.24). A
similarly substituted 4-pyridone ring has been reported in the
recently revised structure of aspernigrin A isolated from Aspergillus
niger and Cladosporium herbarum.24 The NMR signals of this ring
compared favorably with berkeleyamide C (S11, Supporting
Information).
Although the terminal benzyl and isobutyl moieties suggested
that berkeleyamide D (7) belonged to the same family of compounds
as berkeleyamides A-C, the NMR data indicated many structural
differences. Berkeleyamide D (7) had a molecular formula of
C18H21NO5 established by HRESIMS. This formula required nine
units of unsaturation. With only 10 sp2-hybridized carbons it was
clear that 7 contained an additional ring. The benzylic and isobutyl
moieties accounted for C11H16 and four of the nine DBE. The central
portion of the molecule accommodated C7H5NO5 and five DBE.
The presence of an amide was indicated by the IR absorbance
at 1684 cm-1 and by the carbonyl absorbance in the 13C NMR
spectrum at δ 164.1 (Table 1). An IR absorbance at 1736 cm-1
and a carbonyl absorbance of δ 199.4 indicated the presence of a
ketone. 13C NMR data also indicated the presence of two unusually
deshielded oxygen-bearing quaternary carbons (δ 95.3 and 84.9),
an oxygen-bearing methine (δ 75.1), and a highly asymmetric olefin
(δ 104.4, 197.8) influenced by an electron-withdrawing functionality
at one end and an electron-donating group at the other. As the
amide, ketone, and olefin accounted for only three DBE, two fused
rings were required to accommodate the last two DBE. Of the two
methines present in this moiety, one was olefinic C-9 (δC 104.4,
δH 5.35) and the other was oxygen-bearing C-19 (δC 75.1, δH 4.41,
d, J ) 10.0). H-19 showed J-coupling to an -OH proton (δ 3.04,
d, J ) 10.0), indicating that it was a secondary alcohol. This
spectrum also showed proton resonances for NH and/or OH protons
at δ 6.78, bs and 5.48, bs. The construction of the molecule from
these pieces required extensive analysis of the NMR data.
The benzylic methylene C-7 (δ 37.4) provided a convenient
starting point to generate the structure of 7. H2-7 (δ 3.98, d, J )
17.4 and 3.96, d, J ) 17.4) showed HMBC correlations to several
of the aromatic carbons, as well as to olefinic carbons C-8 and C-9
(δ 197.9 and 104.4, respectively). Long-range COSY correlations
could be seen from H2-7 and aromatic H-1 and H-5 to olefinic H-9.
H-9 showed HMBC correlations to the carbons resonating at δ
197.8, 199.4, and 95.3. The C-19-OH, H-19, and H-13 (δ 6.78)
also showed HMBC correlations to quaternary C-11 (δ 95.3).
Hence, only one carbon remained to be assigned. H-19 and all three
of the OH/NH groups showed long-range coupling to quaternary
C-14 (δ 84.9). These connectivity data could be accommodated
by the [C-7–C-14] unit shown in 7. The isobutyl methylene H2-15
(δ 1.88) also showed connectivity to C-14, which completed the
carbon skeleton of compound 7. Important HMBC correlations are
shown in Figure 3.
Experimental Section
General Experimental Procedures. Optical rotations were recorded
on a Perkin-Elmer 241 MC polarimeter using a 1.0 mL cell. UV spectra
1
were recorded on a Hewlett-Packard 8453 spectrophotometer. H and
13C NMR spectra were run on a Bruker DPX-300 spectrometer. H
1
NMR spectra were recorded at 300 MHz, and the 13C NMR spectra
were recorded at 75 MHz unless otherwise noted. All of the chemical
shifts were recorded with respect to the deuterated solvent shift (CDCl3,
δ 7.24 for the proton resonance and δ 77.0 for the carbon). IR spectra
were recorded on a Nicolet NEXUS 670 FT-IR spectrometer. Mass
Berkeleyamide D (7) showed some interesting derivatization
patterns when treated with a variety of silylating agents. When 7

Notes
Journal of Natural Products, 2008, Vol. 71, No. 5 859
Scheme 1. Possible Biosynthetic Pathway for the Production of the Berkeleyamides
spectra were provided by the University of Montana mass spectrometer
facility in Missoula, Montana. HPLC separations were made on a Varian
Dynamax (250 × 21.4 mm) column. All solvents used were spectral
grade.
Berkeleyamide D (7): oil, [R]25 -56.9 (c 0.007, MeOH); UV
D
(MeOH) λmax (log ꢀ) 272 (3.8), 206 (4.1); IR (CHCl3) νmax 3289, 3024,
2961, 2871, 1736, 1684, 1582, 1385, 1155, 1121, 961 cm-1; 1H NMR
and 13C NMR, see Table 1; EIMS m/z (rel) 313 (2), 295 (4), 217 (65),
57 (100); HREIMS m/z 313.1302 [M - 18]+ (calcd for C18H19NO4,
Collection, Extraction, and Isolation Procedures. The collection
and isolation of Berkeley Pit fungi has previously been described.2,3,7
The Berkeley Pit Penicillium rubrum isolate was grown in potato
dextrose broth (26 flasks with 300 mL of broth per flask, acidified to
pH 2.7 with concentrated H2SO4) for 6 days on a shaker table and
then 15 days in still culture. The fungus was then killed with MeOH
(50 mL per flask), mycelia were removed by filtration, and the broth
was extracted with CHCl3 (3 × 1 L). Removal of the CHCl3 in Vacuo
gave 1.13 g of crude extract. This extract showed very good activity
in our enzyme inhibition assays. The crude extract was chromatographed
on a flash Si gel column with a gradient system that began with pure
hexanes to which increasing amounts of isopropyl alcohol were added,
ending with pure isopropyl alcohol. The column was washed with
MeOH. The fraction that eluted with 10% isopropyl alcohol/hexanes
was further purified with silica gel HPLC using an isopropyl alcohol/
hexanes gradient to give berkeleyamide A (1, 15.6 mg), berkeleyamide
B (4, 22.9 mg), berkeleyamide C (5, 5.4 mg), and berkeleyamide D
(7, 20.3 mg).
313.1314); HRESIMS m/z 354.1318 [M
+
Na]+ (calcd for
C18H21NO5Na, 354.1317), 314.1383 [M - 17]+ (calcd for C18H20NO4,
314.1392).
Silylation of Berkeleyamide D (7) with TMSIM. Compound 7 (0.5
mg) was treated with trimethylsilylimidazole (500 µL) and heated for
2 min at 50 °C, and then the mixture was subjected to ESIMS. ESIMS
m/z 570 [C18H21NO5 - 3H + 3(TMS) + Na]+.
Silylation of Berkeleyamide D (7) with BSTFA. Compound 7 (0.5
mg) was treated with N,O-bis(trimethylsilyl)trifluoroacetamide (500 µL)
and heated for 2 min at 50 °C, and then the mixture was subjected to
ESIMS. ESIMS m/z 498 [C18H21NO5 - 2H + 2(TMS) + Na]+.
Growth of Penicillium rubrum with 15N-Labeled Leucine. Peni-
cillium rubrum was grown in acidified potato dextrose broth (20 flasks
with 300 mL of broth per flask and 30 flasks with 700 mL) as described
above. The flasks were grown for 14 days, and 15N leucine (10 mg of
powder to each small flask and 20 mg to each large flask; Cambridge
Isotope Laboratories) was added. The cultures were allowed to grow
for an additional 7 days. The fungus was then killed and extracted with
CHCl3 as described above. Removal of the CHCl3 gave 1.80 g of crude
extract. This extract was fractionated by flash Si gel chromatography
followed by Si gel HPLC using isopropyl alcohol/hexanes mixtures to
give berkeleyamide B (4, 9.6 mg) and berkeleyamide D (7, 5.5 mg).
The ESI mass spectra of these compounds were compared to the spectra
from the unlabeled samples. Berkeleyamide B (4): ESIMS m/z
(intensity) 394.1 [M + Na]+ (100%), 395.1 [M + Na + 1]+ (25.1%),
15N-labeled sample 394.1 (100%), 395.1 (27.0%). Berkeleyamide D
(7): ESIMS m/z (intensity) 354.1 [M + Na]+ (100%), 355.1 [M + Na
+ 1]+ (23.8%), 15N-labeled sample 354.1 (100%), 355.1 (26.0%).
Signal Transduction Assays. MMP-3 colorimetric (AK-400) and
caspase-1 (AK-701) drug discovery kits were used for this assay
(BIOMOL International). Crude extracts were tested at 1000 µg/mL,
and column fractions were tested at 500 µg/mL.
Berkeleyamide A (1): oil, [R]25D -1.0 (c 0.017, MeOH); IR (CHCl3)
1
νmax 3428, 3012, 2960, 1715, 1685, 1368, 1100, 911 cm-1; H NMR
and 13C NMR, see Table 1; HRESIMS m/z 304.1927 [M + H]+ (calcd
for C18H26NO3, 304.1913).
Acetylation of Berkeleyamide A (1). Compound 1 (2.0 mg) was
dissolved in pyridine (50 µL) and Ac2O (50 µL) and stirred for 24 h,
after which the solvents were removed in Vacuo to give 2 as an oil
(2.1 mg): 1H NMR (CDCl3) δ 7.13–7.35 (m, 5 H, aromatics), 5.93 (br
s, NH), 5.48 (q, J ) 6.5 Hz, H-10), 3.71 (s, 2H, H-7), 3.64 (m, H-14),
3.01 (dd, H-9), 2.85 (m, H-9), 2.69 (m, H-11), 2.11 (m, H-19), 1.96 (s,
3H, Ac), 1.68 (m, H-19), 1.54 (m, H-16), 1.21–1.43 (m, 2H, H-15),
0.89 (d, J ) 6.6 Hz, 6 H, H-17, H-18).
Chiral Derivatization of Berkeleyamide A (1). Berkeleyamide A
(1, 1.0 mg) was dissolved in dry pyridine (100 µL), and either the R or
S stereoisomer of R-methoxy-R-trifluoromethylphenylacetyl chloride
(6 µL) was added. The mixtures were stirred for 24 h under N2. MeOH
(400 µL) was added to terminate the reaction, and the solvents were
removed in Vacuo. The reaction mixtures were then each passed through
a small Si gel column and eluted with CH2Cl2 to give the products.
(R)-MTPA ester(3a): 1H NMR (CDCl3) δ 7.32–7.42 (m, 5 H,
aromatics), 5.78 (q, J ) 6.6 Hz, H-10), 5.57 (br s, NH), 3.68 (bs, 2H,
H-7), 3.64 (m, H-14), 2.95 (dd, J ) 16.9, 6.1 Hz, H-9), 2.89 (dd, J )
16.9, 6.7 Hz, H-9), 2.77 (m, H-11), 2.05 (m, H-19), 1.57 (m, H-19),
1.34–1.49 (m, 3H, H-15, H-16), 0.84 (d, J ) 6.7 Hz, 6H, H-17, H-18).
(S)-MTPA ester (3b): 1H NMR (CDCl3) δ 7.32–7.41 (m, 5H,
aromatics), 5.79 (q, J ) 6.1 Hz, H-10), 5.53 (br s, NH), 3.72 (br s, 2H,
H-7), 3.64 (m, H-14), 3.09 (dd, J ) 16.9, 6.9 Hz, H-9), 2.91 (dd, J )
16.9, 6.0 Hz, H-9), 2.67 (m, H-11), 2.04 (m, H-19), 1.54 (m, H-19),
1.34–1.49 (m, 3H, H-15, H-16), 0.85 (d, J ) 6.6 Hz, 6H, H-17, H-18).
Acknowledgment. We thank B. Parker and the University of
Montana for mass spectrometry data. We thank the National Science
Foundation grant CHE-9977213 for acquisition of a NMR spectrometer
and the MJ Murdock Charitable Trust ref no. 99009:JVZ:11/18/99 for
acquisition of the mass spectrometer. The project described was
supported by NIH grant P20 RR16455-04 from the INBRE-BRIN
Program of the National Center for Research Resources and USGS
grant 02HQGR0121. The views and conclusions contained in this
document are those of the authors and should not be interpreted as
representing the official policies, either expressed or implied, of NIH
or of USGS.
1
Supporting Information Available: H and 13C NMR spectra for
Berkeleyamide B (4): oil, [R]25 +39.6 (c 0.023, CHCl3); IR
D
the berkeleyamides A-D (1, 4, 5, 7) and the comparison of selected
NMR data to known compounds are available free of charge via the
Internet at http://pubs.acs.org.
(CHCl3) νmax 3025, 2962, 1754, 1697, 1654, 1616, 1574, 1506, 1411,
1189, 1037, 910 cm-1; 1H NMR and 13C NMR, see Table 1; HRESIMS
m/z 372.1403 [M + H]+ (calcd for C20H22NO6, 372.1369).
Berkeleyamide C (5): oil, [R]25 +24.9 (c 0.007, CHCl3); IR
D
(CHCl3) νmax 3448, 2955, 2866, 1735, 1681, 1634, 1502, 1215, 1033
References and Notes
1
cm-1; H NMR and 13C NMR, see Table 1; HRESIMS m/z 415.1870
[M + H]+ (calcd for C22H27N2O6, 415.1869).
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1
(2.1 mg): H NMR (CDCl3) δ 13.23 (bs, NH), 8.44 (bs, 1H, H-19),
7.26–7.42 (m, 5H, aromatics), 6.84 (s, 1H, H-9), 6.83 (s, 1H, H-7),
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